ABSTRACT
BACKGROUND: SIN3 (SWI-Independent) is part of a transcriptional deacetylase complex, which generally mediates the formation of repressive chromatin. The purpose of this work was to study possible interactions between corepressors human SIN3B (hSIN3B) and the ETO homologues - ETO (eight twenty-one), MTG16 (myeloid-transforming gene 16) and MTGR1 (MTG-related protein 1). In addition, the subnuclear localization of the hSIN3B and the ETO homologues was also examined. RESULTS: A ubiquitous expression of hSIN3B was observed in adult and fetal tissues. Results with both ectopically expressed proteins in COS-7 cells and endogeneous proteins in the K562 human erytholeukemia cell line demonstrated interactions between hSIN3B and ETO or MTG16 but not MTGR1. Furthermore, nuclear extract of primary placental cells showed complexes between hSIN3B and ETO. The interaction between hSIN3B and ETO required an intact amino-terminus of ETO and the NHR2 domain. A nucleolar localization of hSIN3B and all the ETO homologues was demonstrated upon overexpression in COS-7 cells, and confirmed for the endogeneously expressed proteins in K562 cells. However, hSIN3B did not colocalize or interact with the leukemia-associated AML1 -ETO. CONCLUSION: Our data from protein-protein interactions and immunolocalization experiments support that hSIN3B is a potential member of a corepressor complex involving selective ETO homologues.
Subject(s)
Cell Nucleolus/metabolism , DNA-Binding Proteins/metabolism , Leukemia/metabolism , Proto-Oncogene Proteins/metabolism , Repressor Proteins/metabolism , Transcription Factors/metabolism , Animals , Base Sequence , COS Cells , Cell Line , Chlorocebus aethiops , DNA Primers/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Female , Humans , K562 Cells , Leukemia/genetics , Multiprotein Complexes , Phosphoproteins/genetics , Phosphoproteins/metabolism , Placenta/metabolism , Pregnancy , Protein Structure, Tertiary , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , RUNX1 Translocation Partner 1 Protein , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Repressor Proteins/genetics , Tissue Distribution , Transcription Factors/chemistry , Transcription Factors/genetics , Transfection , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolismABSTRACT
The Eight twenty-one (ETO) homologues are nuclear repressor proteins including ETO, myeloid-transforming gene-related protein 1 (MTGR1), and myeloid-transforming gene chromosome 16 (MTG16). ETO and MTG16 are both part of fusion proteins resulting from chromosomal translocations associated with acute myeloid leukemia. Expression of these chimeras results in a differentiation block that contributes to the onset of leukemia. In order to elucidate the relation between the ETO homologues and hematopoietic differentiation, we determined the expression of the homologues during differentiation of leukemic and normal hematopoietic cells. Our results showed MTGR1 and MTG16 to be ubiquitously expressed in leukemic cell lines, whereas expression of ETO was observed only in an erythroleukemic cell line. The MTGR1 and MTG16 proteins decreased during all trans-retinoic acid-, but not vitamin D(3)-induced differentiation of leukemic cells. The reduction seemed to reflect a decrease in transcript levels as well as in protein stability. MTGR1 transcripts were ubiquitously expressed in human bone marrow cells. The MTG16 transcripts of CD34(+) progenitor cells were rapidly downregulated by cytokine-induced differentiation into myeloid or erythroid lineages. ETO transcripts, present at very low abundance in CD34(+) progenitor cells, were transiently upregulated during erythroid differentiation. In conclusion, the differential expression of the ETO homologues suggests that they may have a potential role in hematopoietic differentiation.
Subject(s)
DNA-Binding Proteins/genetics , Hematopoietic Stem Cells/cytology , Proto-Oncogene Proteins/genetics , Transcription Factors/genetics , Bone Marrow Cells/cytology , Cell Differentiation , Cell Line , Cell Line, Tumor , DNA Primers , Flow Cytometry , Gene Expression Regulation, Neoplastic/drug effects , HL-60 Cells , Humans , K562 Cells , Kidney , Leukemia , Phosphoproteins/genetics , Polymerase Chain Reaction , Repressor Proteins/genetics , Reverse Transcriptase Polymerase Chain Reaction , Tetradecanoylphorbol Acetate/pharmacology , Tretinoin/pharmacology , Tumor Suppressor Proteins/genetics , U937 CellsABSTRACT
The eight-twenty-one (ETO) homologues, represented by ETO, myeloid transforming gene-related protein 1 (MTGR1) and myeloid transforming gene chromosome 16 (MTG16), are nuclear repressor proteins. ETO is part of the fusion protein acute myeloid leukaemia (AML)1-ETO, resulting from the translocation (8;21). Similarly, MTG16 is disrupted to become part of AML1/MTG16 in t(16;21). The aberrant expression of these chimeras could affect interplay between ETO homologues and contribute to the leukaemogenic process. We investigated possible interactions between the ETO homologues. Ectopic co-expression in COS-cells resulted in heterodimerisation of the various ETO homologues suggesting that they may co-operate. Similarly, the chimeric oncoprotein AML1-ETO interacted with both MTGR1 and MTG16. However, results from cell lines endogenously expressing more than one ETO homologue did not demonstrate co-precipitation. Results from IP-Western and size determination by gel filtration of deletion mutants expressed in COS-cells, indicated an important role of the HHR domain for oligomerisation. A role was also suggested for the Nervy domain in the homologue interactions. Our results suggest that ETO homologues can interact with each other as well as with AML1-ETO, although it is unclear as to what extent these interactions occur in vivo.
